SUMMARY One of the most common problems in clinical orthopaedics continues to be ?aseptic loosening? due to inflammatory osteolysis. Wear particles from the implants stimulate macrophages to produce inflammatory cytokines, which induce local osteolysis and inhibit osteogenic repair of the osteolysis. Our long-term goal is to discover novel underlying mechanisms and thereby identify novel therapeutic targets for patients with ?aseptic loosening?. This R21 A1 application will test the overall hypothesis that extracellular ATP (eATP) increases the biologic activity of orthopaedic wear particles. Consistent with the overall hypothesis, our preliminary results indicate that wear particles stimulate macrophages to release ATP. eATP is the only known ligand for P2X7R, which is the primary macrophage receptor for elevated levels of eATP. Stimulation of P2X7R by eATP increases some types of inflammation but has not previously been studied in ?aseptic loosening?. Aim 1 (proof-of-principle aim) will therefore determine how eATP and P2X7R affect the biologic activity of wear particles. Stimulation of the P2X7R by eATP can activate the NLRP3 inflammasome and the NLRP3 inflammasome is primarily responsible for processing pro-IL1 and pro-IL18 to the active cytokines in response to wear particles. However, the NLRP3 inflammasome must be primed prior to activation. Our preliminary results suggest that wear particles can both prime and activate the NLRP3 inflammasome. We previously showed that the adherence of bacterial pathogen-associate molecular patterns (PAMPs) substantially increases the biologic activity of the particles. Aim 2 (mechanistic aim) will therefore determine how wear particles (with and without adherent PAMPs) affect ATP release, the P2X7R, and the NLRP3 inflammasome. Highly-specific, drug-like antagonists of P2X7R can reduce inflammation in animal models and are in clinical trials for other inflammatory conditions. Aim 3 (translational aim) will therefore determine the effects of antagonists of P2X7R on biologic activity of the wear particles. This project is ideal for the R21 mechanism: It is high risk because there are no previous studies on the effects of eATP or P2X7R in ?aseptic loosening?. It has the potential for high impact because the P2X7R antagonists were well tolerated in clinical trials for other inflammatory conditions. Our team possesses the necessary expertise and thus is uniquely positioned to efficiently complete this project.